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result(s) for
"cleavage"
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Pre-mRNA processing factors differentially impact coordination between co-transcriptional cleavage and transcription termination
Co-transcriptional cleavage and transcription termination are closely related processes during mRNA maturation, yet their coordination remains poorly understood due to difficulties in detecting these transient events. Here, we applied single-molecule nascent RNA sequencing to simultaneously capture the cleavage status and readthrough distance on the same nascent RNA molecules and characterize 14 mutants of various pre-mRNA processing factors in
Arabidopsis
. Our results reveal diverse roles for these processing factors in coordinating cleavage and termination: core components of CPSF and CstF complex stimulate both cleavage and termination, facilitating access to exoribonuclease AtXRN3; mutations in nuclear poly(A) polymerase PAPS1 and AtXRN3 caused delayed termination with minimal effects on cleavage, suggesting their roles are further downstream; BORDER proteins facilitate termination while simultaneously inhibiting cleavage, suggesting a complex interplay between these two actions; the phosphatase SSU72 specifically promotes efficient termination without affecting cleavage. Our method also enables us to distinguish cleaved readthrough transcripts from full-length readthrough, and we found termination factor FPA specifically promotes termination of cleaved readthrough, suggesting FPA facilitates access of AtXRN3 to the 3’ cleavage product. Our comprehensive datasets reveal cleavage and termination are highly coordinated during pre-mRNA processing.
This study reveals how pre-mRNA processing factors in
Arabidopsis
distinctly coordinate cleavage and transcription termination, highlighting complex roles of CPSF, CstF, PAPS1, BORDER proteins, and FPA in efficient mRNA maturation.
Journal Article
Direct Unequal Cleavages: Embryo Developmental Competence, Genetic Constitution and Clinical Outcome
To investigate the prevalence, developmental potential, chromosomal constitution and clinical outcome of embryos with direct unequal cleavages (DUC).
A retrospective observational study.
Academic Institution.
21,261 embryos from 3,155 cycles cultured in EmbryoScope®.
The total incidence of DUCs per embryo occupying the first three cleavages were 26.1%. Depending of the cell stage, DUC rate was 9.8% at first cleavage (DUC-1), 9.1% at second cleavage (DUC-2), and 3.7% at third cleavage (DUC-3) with 3.6% of embryos exhibiting multiple DUCs (DUC-Plus). The occurrence of DUCs was not correlated with female gamete age or source. The incidence of DUC-1 was significantly higher in embryos fertilized by epididymal and testicular sperm (13.6% and 11.4%, respectively) compared to ejaculated sperm (9.1%, all p<0.05). The total incidences of DUCs were strongly correlated with the onset of blastomere multinucleation (MNB) during the first three divisions. In MNB embryos, DUCs incidence are two to three times more likely to develop when compared to non-MNB embryos (OR = 3.11, 95% CI [2.64, 3.67] at 1-cell stage, OR = 2.64, 95% CI [2.39, 2.91] at 2-cell stage and OR = 2.51, 95% CI [1.84, 3.43] at 4-cell stage). The blastocyst formation rates gradually decreased from 61.0% in non-DUC to 40.2% in DUC-3, 18.8% in DUC-2, 8.2% in DUC-1 and 5.6% in multiple DUC embryos (DUC-Plus). The known implantation rates (FH) for day 3 (D3) transfers were 12.42% (n = 3172) in Non-DUC embryos, 6.3% (n = 127) in DUC-3, and 2.7% (n = 260) in DUC-2 embryos. No live births resulted from either DUC-1 (n = 225) or DUC-Plus (n = 100) embryo transfers. For blastocyst transfers, lower implantation rates (33.3%) but similar live birth (LB) rates (40%) were observed if DUC blastocysts were transferred. Comparatively rates in Non-DUC blastocyst were 45.2% and 34.8%, respectively. The euploid rate gradually increased from DUC-1, -2, -3 to Non-DUC (13.3%, 19.5%, 33.3%, 45.6%, p<0.001) for D3 biopsied embryos. Interestingly, the trend of decreased euploidy disappeared in DUC D5/6 biopsied embryos and similar rates were exemplified in DUC (D5 56.3%, D6 35.6%) vs. non-DUC (D5 51.4%, D6 33.8%) embryos.
Blastocyst formation, implantation potential and euploid rate were significantly reduced in DUC embryos. DUC embryos should be deselected for D3 transfers, but should be culture to blastocyst stage for possible ET.
Journal Article
Structure of an active human histone pre-mRNA 3′-end processing machinery
The 3′-end processing machinery for metazoan replication-dependent histone precursor messenger RNAs (pre-mRNAs) contains the U7 small nuclear ribonucleoprotein and shares the key cleavage module with the canonical cleavage and polyadenylation machinery. We reconstituted an active human histone pre-mRNA processing machinery using 13 recombinant proteins and two RNAs and determined its structure by cryo–electron microscopy. The overall structure is highly asymmetrical and resembles an amphora with one long handle. We captured the pre-mRNA in the active site of the endonuclease, the 73-kilodalton subunit of the cleavage and polyadenylation specificity factor, poised for cleavage. The endonuclease and the entire cleavage module undergo extensive rearrangements for activation, triggered through the recognition of the duplex between the authentic pre-mRNA and U7 small nuclear RNA (snRNA). Our study also has notable implications for understanding canonical and snRNA 3′-end processing.
Journal Article
A potent antimalarial benzoxaborole targets a Plasmodium falciparum cleavage and polyadenylation specificity factor homologue
Benzoxaboroles are effective against bacterial, fungal and protozoan pathogens. We report potent activity of the benzoxaborole AN3661 against
Plasmodium falciparum
laboratory-adapted strains (mean IC
50
32 nM), Ugandan field isolates (mean
ex vivo
IC
50
64 nM), and murine
P. berghei
and
P. falciparum
infections (day 4 ED
90
0.34 and 0.57 mg kg
−1
, respectively). Multiple
P. falciparum
lines selected
in vitro
for resistance to AN3661 harboured point mutations in
pfcpsf3
, which encodes a homologue of mammalian cleavage and polyadenylation specificity factor subunit 3 (CPSF-73 or CPSF3). CRISPR-Cas9-mediated introduction of
pfcpsf3
mutations into parental lines recapitulated AN3661 resistance. PfCPSF3 homology models placed these mutations in the active site, where AN3661 is predicted to bind. Transcripts for three trophozoite-expressed genes were lost in AN3661-treated trophozoites, which was not observed in parasites selected or engineered for AN3661 resistance. Our results identify the pre-mRNA processing factor PfCPSF3 as a promising antimalarial drug target.
Benzoxaboroles have been shown to be active against different pathogens. Here, the authors show that the benzoxaborole AN3661 inhibits
Plasmodium falciparum in vitro
and in mouse models, and identify a homologue of a mammalian cleavage and polyadenylation specificity factor as a drug target.
Journal Article
Polyadenylation factor CPSF-73 is the pre-mRNA 3'-end-processing endonuclease
Most eukaryotic messenger RNA precursors (pre-mRNAs) undergo extensive maturational processing, including cleavage and polyadenylation at the 3'-end. Despite the characterization of many proteins that are required for the cleavage reaction, the identity of the endonuclease is not known. Recent analyses indicated that the 73-kDa subunit of cleavage and polyadenylation specificity factor (CPSF-73) might be the endonuclease for this and related reactions, although no direct data confirmed this. Here we report the crystal structures of human CPSF-73 at 2.1 A resolution, complexed with zinc ions and a sulphate that might mimic the phosphate group of the substrate, and the related yeast protein CPSF-100 (Ydh1) at 2.5 A resolution. Both CPSF-73 and CPSF-100 contain two domains, a metallo-beta-lactamase domain and a novel beta-CASP (named for metallo-beta-lactamase, CPSF, Artemis, Snm1, Pso2) domain. The active site of CPSF-73, with two zinc ions, is located at the interface of the two domains. Purified recombinant CPSF-73 possesses RNA endonuclease activity, and mutations that disrupt zinc binding in the active site abolish this activity. Our studies provide the first direct experimental evidence that CPSF-73 is the pre-mRNA 3'-end-processing endonuclease.
Journal Article
CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity
CRISPR techniques are allowing the development of technologies for nucleic acid detection (see the Perspective by Chertow). Taking advantages of the distinctive enzymatic properties of CRISPR enzymes, Gootenberg et al. developed an improved nucleic acid detection technology for multiplexed quantitative and highly sensitive detection, combined with lateral flow for visual readout. Myhrvold et al. added a sample preparation protocol to create a field-deployable viral diagnostic platform for rapid detection of specific strains of pathogens in clinical samples. Cas12a (also known as Cpf1), a type V CRISPR protein, cleaves double-stranded DNA and has been adapted for genome editing. Chen et al. discovered that Cas12a also processes single-stranded DNA threading activity. A technology platform based on this activity detected human papillomavirus in patient samples with high sensitivity. Science , this issue p. 439 , p. 444 , p. 436 ; see also p. 381 Single-stranded DNase activity upon guide RNA–dependent DNA binding can be harnessed for rapid and specific nucleic acid detection. CRISPR-Cas12a (Cpf1) proteins are RNA-guided enzymes that bind and cut DNA as components of bacterial adaptive immune systems. Like CRISPR-Cas9, Cas12a has been harnessed for genome editing on the basis of its ability to generate targeted, double-stranded DNA breaks. Here we show that RNA-guided DNA binding unleashes indiscriminate single-stranded DNA (ssDNA) cleavage activity by Cas12a that completely degrades ssDNA molecules. We find that target-activated, nonspecific single-stranded deoxyribonuclease (ssDNase) cleavage is also a property of other type V CRISPR-Cas12 enzymes. By combining Cas12a ssDNase activation with isothermal amplification, we create a method termed DNA endonuclease-targeted CRISPR trans reporter (DETECTR), which achieves attomolar sensitivity for DNA detection. DETECTR enables rapid and specific detection of human papillomavirus in patient samples, thereby providing a simple platform for molecular diagnostics.
Journal Article
miRNA-dependent gene silencing involving Ago2-mediated cleavage of a circular antisense RNA
MicroRNAs (miRNAs) are ∼22 nt non‐coding RNAs that typically bind to the 3′ UTR of target mRNAs in the cytoplasm, resulting in mRNA destabilization and translational repression. Here, we report that miRNAs can also regulate gene expression by targeting non‐coding antisense transcripts in human cells. Specifically, we show that miR‐671 directs cleavage of a circular antisense transcript of the Cerebellar Degeneration‐Related protein 1 (
CDR1
) locus in an Ago2‐slicer‐dependent manner. The resulting downregulation of circular antisense has a concomitant decrease in
CDR1
mRNA levels, independently of heterochromatin formation. This study provides the first evidence for non‐coding antisense transcripts as functional miRNA targets, and a novel regulatory mechanism involving a positive correlation between mRNA and antisense circular RNA levels.
Natural antisense transcripts appear to have widespread roles in gene regulation. This study provides the first example of miRNA targeting of an antisense transcript. Nuclear miR‐671 targets and cleaves a circular antisense transcript expressed from the CDR1 locus, reducing CDR1 mRNA levels.
Journal Article
Rationally engineered Cas9 nucleases with improved specificity
The RNA-guided endonuclease Cas9 is a versatile genome-editing tool with a broad range of applications from therapeutics to functional annotation of genes. Cas9 creates double-strand breaks (DSBs) at targeted genomic loci complementary to a short RNA guide. However, Cas9 can cleave off-target sites that are not fully complementary to the guide, which poses a major challenge for genome editing. Here, we use structure-guided protein engineering to improve the specificity of Streptococcus pyogenes Cas9 (SpCas9). Using targeted deep sequencing and unbiased whole-genome off-target analysis to assess Cas9-mediated DNA cleavage in human cells, we demonstrate that \"enhanced specificity\" SpCas9 (eSpCas9) variants reduce off-target effects and maintain robust on-target cleavage. Thus, eSpCas9 could be broadly useful for genome-editing applications requiring a high level of specificity.
Journal Article
Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases
Programmable genome integration of large, diverse DNA cargo without DNA repair of exposed DNA double-strand breaks remains an unsolved challenge in genome editing. We present programmable addition via site-specific targeting elements (PASTE), which uses a CRISPR–Cas9 nickase fused to both a reverse transcriptase and serine integrase for targeted genomic recruitment and integration of desired payloads. We demonstrate integration of sequences as large as ~36 kilobases at multiple genomic loci across three human cell lines, primary T cells and non-dividing primary human hepatocytes. To augment PASTE, we discovered 25,614 serine integrases and cognate attachment sites from metagenomes and engineered orthologs with higher activity and shorter recognition sequences for efficient programmable integration. PASTE has editing efficiencies similar to or exceeding those of homology-directed repair and non-homologous end joining-based methods, with activity in non-dividing cells and in vivo with fewer detectable off-target events. PASTE expands the capabilities of genome editing by allowing large, multiplexed gene insertion without reliance on DNA repair pathways.
Large sequences are integrated site specifically into the human genome without double-strand DNA cleavage.
Journal Article